Intratype variants of the E2 protein from human papillomavirus type 18 induce different gene expression profiles associated with apoptosis and cell proliferation

Abstract

Persistent infections with high-risk human papillomaviruses (HR-HPVs) are linked to the development of cervical cancer due to a deregulation of the productive viral cycle in the host cell, leading to cell transformation. The E2 viral protein is expressed early during an HPV infection and regulates viral replication and transcription. Other functions have been attributed to E2, such as the promotion of apoptosis that are independent of its role in the regulation of the expression of E6 and E7 viral oncogenes. Moreover, it has been shown that the HPV16 E2 protein has regulatory effects on cellular gene expression, suggesting that it participates in the modulation of different cellular processes. Intratype genomic variations within high-risk HPV types have an impact on the prognosis of HPV-related lesions. Nevertheless, the biological significance of HPV18 E2 intratype variations has not been analysed previously. The aim of this study was to determine whether HPV18 E2 intratype variations differentially modulate gene expression and whether cell-death-related genes are affected by variations in E2. We demonstrate that HPV18 E2 intratype Asian Amerindian (AsAi) and African (Af) variants differentially affect gene expression profiles. Although the E2-AsAi variant was found to modulate a larger number of cellular genes, both E2 variants affected similar cellular processes. Nevertheless, E2-AsAi and E2-Af variants showed differences in their ability to induce apoptosis, where E2-Af had a stronger effect. The differences in gene expression profiles in cells harbouring E2 intratype variants suggest a possible effect on diverse cellular signalling pathways, and this might suggest an approach for identifying biological processes regulated by HPV18 E2 intratype variants.

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Change history

  • 18 May 2019

    The Given names of the author Alma Mariana Fuentes-González was incorrectly tagged in original publication and corrected here. The original article has been corrected.

Abbreviations

HPV:

Human papillomavirus

HR:

High-risk

CC:

Cervical cancer

DMEM:

Dulbecco´s modified Eagle´s medium

RT-qPCR:

Quantitative reverse transcription PCR

ΔΔ Ct:

Double delta Ct

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Acknowledgements

Alma Mariana Fuentes-González is a Ph.D. student from Programa de Doctorado en Ciencias Biomédicas, Universidad Nacional Autónoma de México (UNAM) and received a scholarship from CONACyT (268404). We thank María Alexandra Rodríguez-Sastre and Patricia de la Torre from Instituto de Investigaciones Biomédicas, UNAM for technical assistance.

Funding

This work was partially supported by CONACYT México grant CB-166808 and INCan Ref. 005/017/IBI.

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Contributions

All of the authors listed made substantial contributions to the manuscript and qualify for authorship, and no authors have been omitted. Conception and design, ML; development of methodology and acquisition of data, AMFG, JOMB, JFR and JMM; analysis and interpretation of data, AMFG, JMM, JOMB, ML, APT, CPP; writing and revision of the manuscript, AMFG, JMM, ML, JOMB and ACP. All the authors read and approved the final manuscript.

Corresponding author

Correspondence to Marcela Lizano.

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Conflict of interest

There are no commercial or financial conflicts of interest to declare.

Additional information

The original version of this article was revised: The Given\Family names of the author Alma Mariana Fuentes‑González was incorrectly assigned in original publication.

Handling Editor: Zhongjie Shi.

Electronic supplementary material

Below is the link to the electronic supplementary material.

Figure S1.

Nucleotide and amino acid changes between HPV18 E2 intratype variants. Differences of nucleotide and amino acid positions within HPV18 E2-AsAi and HPV18 E2-Af are shown. Del: deleted nucleotide (JPEG 100 kb)

Figure S2.

Cell death and DNA damage response genes regulated by HPV18 E2-AsAi and E2-Af. A) HPV18 E2-AsAi and B) HPV18 E2-Af regulate the expression of genes associated to cell death and DNA damage response illustrated by the Ingenuity Pathway Analysis Tool. Up-regulated genes are shown in red and down-regulated genes are depicted in blue (JPEG 998 kb)

Supplementary Table S1.

Sequences of primers used for RT-qPCR assay. 18S, FOSB, EPS15, CASP4, EPHA2, RAB4B, TRAF1, and HPV18 E2 primers were used to validate the expression of the selected genes (DOCX 15 kb)

Supplementary Table S2.

Genes modulated by the HPV18 E2-AsAi variant. After normalization process, gene selection was performed considering a p value ≤ 0.05 and a fold change ± 2.0. Genes differentially expressed compared to the control vector (EV) are listed. Genes with fold change values ≥ 2.0 were considered as up-regulated, while those with fold change values ≤ -2.0 were considered as down-regulated (XLSX 90 kb)

Supplementary Table S3.

Genes modulated by the HPV18 E2-Af variant. After normalization process, gene selection was performed considering a p value ≤ 0.05 and a fold change ± 2.0. Genes differentially expressed compared to the control vector (EV) are listed. Genes with fold change values ≥ 2.0 were considered as up-regulated, while those with fold change values ≤ -2.0 were considered as down-regulated (XLSX 23 kb)

Supplementary Table S4.

Genes up-regulated and down-regulated by HPV18 E2-AsAi involved in death pathways. Analysis performed by the Ingenuity Pathway Analysis Tool shows that from a total of 100 genes associated to apoptotic pathways, 42 were found up-regulated and 58, down-regulated in E2-AsAi expressing cells (XLSX 15 kb)

Supplementary Table S5.

Genes up- and down-regulated by HPV18 E2-Af involved in death pathways. Analysis performed by the Ingenuity Pathway Analysis Tool showed 8 genes up-regulated and 8 down-regulated that are related to cellular death pathways in cells transfected with E2-Af variant (XLSX 10 kb)

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Fuentes-González, A.M., Muñoz-Bello, J.O., Manzo-Merino, J. et al. Intratype variants of the E2 protein from human papillomavirus type 18 induce different gene expression profiles associated with apoptosis and cell proliferation. Arch Virol 164, 1815–1827 (2019). https://doi.org/10.1007/s00705-018-04124-6

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